Wheat germ agglutinin conjugated poly dl lactic co glycolic acid (PLGA) nanoparticles for enhanced uptake and retention of paclitaxel by colon cancer cells

POLY-DL-LACTIC-CO-GLYCOLIC ACID PLGA NANOPARTICLES FOR ENHANCED UPTAKE AND RETENTION OF PACLITAXEL BY COLON CANCER CELLS... The purpose of this project was to confirm the hypothesis tha

POLY-DL-LACTIC-CO-GLYCOLIC ACID (PLGA)

NANOPARTICLES FOR ENHANCED UPTAKE AND

RETENTION OF PACLITAXEL BY COLON CANCER CELLS

Paul, Ho Chi Lui and Lim Lee Yong for their precious guidance, helpful advice and enormous support and patience during my PhD study Their enthusiasm and originality in research will inspire and benefit me the whole life Without them this thesis would not have been possible

Thanks to Department of Pharmacy, National University of Singapore for providing the financial support for me to pursue my PhD degree and thanks to the PhD committee for their precious time to read the thesis Sincere gratitude is also expressed to all the lab officers, including Swee Eng, Sek Eng, Mr Tang, Tang Booy, Josephine and Madam Loy for their technical help and support

To all my friends, past and present, especially Huang Meng, Haishu, Yupeng, Siok Lam, Hanyi, Weiqiang, Dahai, Ma Xiang, Yang Hong, Shili, Wang Zhe, Tarang Thank you for your support, discussions, meetings, outings and jokes

To my parents, my husband, my son, my sisters, thank you for your patience, encouragement, selfless support and putting up with all my frustration and emotion during the journey of my study all along

ACKNOWLEDGEMENTS Ⅰ TABLE OF CONTENTS Ⅱ SUMMARY II

LIST OF TABLES II

LIST OF FIGURES III LIST OF ABBREVIATIONS XVII LIST OF PUBLICATIONS XIX

2.4.2 Lectin blot analysis 55

Chapter 3 Uptake and cytotoxicity of wheat germ agglutinin in colon cell

3.4.1 In vitro cytotoxicity profile of WGA against colon cell lines 69

3.4.3 Laser scanning confocal photomicrographs 75

3.6 Conclusion 83

Chapter 4 Evaluation of anticancer activity of wheat germ agglutinin

4.2 Materials 89

4.3.1 Preparation of WGA-conjugated, paclitaxel-loaded PLGA nanoparticles 90

4.3.2.1 Particle size, zeta potential and morphology 94

4.3.2.2 WGA loading efficiency 95

4.3.2.3 Determination of paclitaxel loading efficiency 96

4.3.2.4 In vitro drug release 96

4.3.3 In vitro cytotoxicity of blank WGA-conjugated PLGA nanoparticles 97 4.3.4 Uptake of blank fWGA (fWN) and fBSA- (fBN) conjugated PLGA nanoparticles 98 4.3.5 Antiproliferation activity of paclitaxel 99 4.3.6 Cellular accumulation and efflux of paclitaxel 100

4.3.7 Visualization of cell-associated nanoparticles 102

4.3.8 Cell morphological and nucleus fragmentation examination 103

4.3.9 Cell cycle analysis by flow cytometry 104

4.3.10 Cellular trafficking of WNP 106

4.3.11 Statistical analysis 107

4.4.1.1 Particle size, zeta potential and morphology 107

4.4.1.2 WGA loading efficiency 109

4.4.1.3 Determination of paclitaxel loading efficiency 109

4.4.1.4 In vitro drug release 110

4.4.2 In vitro cytotoxicity profile of blank WGA-conjugated PLGA nanoparticles 110 4.4.3 Uptake of blank fWGA-conjugated PLGA nanoparticles 113 4.4.4 Antiproliferation activity of paclitaxel 115 4.4.5 Antiproliferation activity of paclitaxel-loaded PLGA nanoparticles 117

4.4.6 Cellular accumulation and efflux of paclitaxel 121 4.4.7 Visualization of cell-associated nanoparticles 123 4.4.8 Cell morphology 125

4.4.9 Cell cycle analysis 128 4.4.10 Cellular trafficking of WGA-conjugated PLGA nanoparticles 131 4.5 Discussion 136 4.6 Conclusion 142 Chapter 5 Effect of mucin on the uptake of nanoparticles 144

5.3.1 Cell culture 150 5.3.2 Alcian blue (AB) and periodic acid Schiff (PAS) staining 151

5.3.9 Visualization of fWNP uptake by LS174T cells 156

5.4.1 Alcian blue (AB) and periodic acid Schiff (PAS) staining 156

5.4.3 In vitro cytotoxicity profile of WGA against LS174T 158

5.4.5 Antiproliferation activity of paclitaxel-loaded nanoparticles 160 5.4.6 Cellular uptake and efflux of paclitaxel 161

5.4.8 Visualization of fWNP uptake by LS174T cells 165

Chapter 6 Conclusions 173

Chapter 7 Future directions 181 Chapter 8 References 185

healthy cells Most current anticancer drugs cannot greatly differentiate between cancerous and normal cells This leads to systemic toxicity and adverse effects Targeted delivery of anticancer agents could offer a more efficient and less harmful solution to overcome this drawback The purpose of this project was to confirm the hypothesis that conjugation of WGA to PLGA nanoparticles loaded with paclitaxel (WNP) could improve the delivery of paclitaxel to colonic cancer cells

Glycosylation patterns of representative colon cancer cells (Caco-2 and HT-29 cells) and normal cells (colon fibroblasts, CCD-18Co cells) were first investigated Our results confirmed the higher expression levels of WGA-binding glycoproteins (N-acetylglucosamine and sialic acid) in the Caco-2 and HT-29 cells, than in the CCD-18Co cells Most of the WGA-recognizable glycoproteins in the Caco-2 and HT-29 cells had molecular weight > 75 kDa, whereas the CCD-18Co cells showed an apparent lack of expression of such large proteins The ranking order of expression of WGA-recognizable glycoproteins in the three cell lines was HT-29 > Caco-2 > CCD-18Co cells

In vitro cytotoxicity and cellular uptake studies were then carried out to evaluate the

potential of WGA for targeted activity against the colon cell models WGA exhibited

Cellular uptake of fWGA ranked in the order of Caco-2 > HT-29 > CCD-18Co cells The higher binding of fWGA to the malignant cells may be associated with the greater expression of WGA-recognizable glycoproteins in these cells relative to the colon fibroblast cells

WGA was conjugated onto the surface of paclitaxel-loaded PLGA nanoparticles to prepare the WNP formulation Cellular uptake and cytotoxicity of WNP were

evaluated in the three colon cell lines In vitro anti-proliferation studies suggested that

the incorporation of WGA enhanced the cytotoxicity of the paclitaxel-loaded PLGA nanoparticles against the cancerous Caco-2 and HT-29 cells Paclitaxel uptake from the WNP formulation at 2h incubation was the highest in the Caco-2 cells compared

to the other two cell lines Caco-2 and HT-29 cells showed preferential uptake of WNP compared to PNP, suggesting that WGA conjugation to the PLGA nanoparticles was advantageous in facilitating the nanoparticle uptake by the cultured colon cancer cells The greater efficacy of WNP correlated well with the higher cellular uptake and sustained intracellular retention of paclitaxel associated with the formulation, which again might be attributed to the over-expression of N-acetyl-D-glucosamine- containing glycoprotein on the colon cell surface About 30% of the endocytosed WNP was observed in the late endo-lysosomes, but fluorimetric measurements

To evaluate the effect of mucin glycoprotein on the cellular uptake of WNP, the uptake experiments were repeated using a mucin-secreting cell line, LS174T The LS174 cells showed no significant differences in paclitaxel uptake from the WNP formulation on day 3 of culture compared to day 6 of culture, despite the significantly higher production of mucin by day 6 The presence of mucin may therefore not be a barrier for the cellular uptake of WGA-conjugated nanoparticles FRAP results showed that fWNP was capable of diffusing in the mucin layer, although the diffusion rate was slowed down by the viscous mucin This diffusion capability allowed the fWNP to remain mobile in the mucin layer and enabled the particles to be transported into the cells Interaction between the mucin glycoprotein and WGA is postulated to serve as a bridge between WNP and the colon cancer cells under the mucin layer

On the basis of these results, it may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer

Table Page

1.1 Microtubule-targeted drugs, their binding sites on tubulin, and their

stages of clinical development 5

1.2 Examples of polymeric nanoparticles in clinical development 16

4.1 Size and zeta potential of PLGA nanoparticles before and after conjugation

with WGA (Data represent mean ± SD, n=3) 108

4.2 IC50 values of paclitaxel formulated as WNP, PNP and P/CreEL IC50

values were evaluated after 24 and 72 h exposure and the results represent

Mean ± SD values (µg/ml) of three independent experiments, each

performed in triplicate ‘*’ means statistical significance compared to

P/CreEL, ‘^’ means statistical significance between the WNP and PNP

formulations 120

4.3 Colocalisation of FITC-WNP (green) and endosome/lysosome (red) in

Caco-2 cells Colocalisation percentage is the percentage of voxels which

have both red and green intensities above threshold, expressed as a percentage

of the total mumber of pixels in the image 134

5.1 Mobile fraction and half-time to steady state fluorescence obtained from the

FRAP plots for fWGA, fWNP and fBSA-NP particles in PBS and the mucin

layer of LS174T samples (Mean ± SD, n=4) 165

1.1 Molecular structure of paclitaxel 7

1.2 Chemical structure of PLGA and its degradation products 19

1.3 Schematic drawing of steps involved in cytosolic delivery of therapeutics

using polymeric nanoparticles (NPs) (1) Cellular association of NPs, (2)

Internalization of NPs into the cells by endocytosis, (3) Endosomal escape

of NPs, (4) Release of therapeutic in cytoplasm, (5) Cytosolic transport of

therapeutic agent, (6) Degradation of drug either in lysosomes or in cytoplasm,

(7) Exocytosis of NPs 26

1.4 Structure of acetylglucosamine–wheat germ agglutinin complex: red,

N-acetylglucosamine; blue / green, wheat germ agglutinin 29

1.5 Possible pathways for lectin-mediated drug delivery to enterocyte as

exemplified by WGA 30

2.1 Lectin blot Scheme Biotinylated WGA attaches to the glycoprotein,

the biotin label is amplified with avidin, and the complex is visualized

with chemiluminescence 50

2.2 Mem-PER reagent protocol 52

2.3 Lectin blot analysis of (a) cell membrane proteins and (b) intracellular

proteins in Caco-2 (Lane 1), HT-29 (Lane 2) and CCD-18Co (Lane 3) cells 56 3.1 Biotransformation of MTT to formazan in cells 64

3.2 In vitro cytotoxicity profiles of WGA against (A) Caco-2; (B) HT-29

and (C) CCD-18Co cells WGA was applied at loading concentrations

of 0.1, 1, 5, 10, 50, 100 and 200 μg/ml for periods ranging from 4 to

72h Cell viability determined by the MTT assay was expressed as a

percent of that obtained for cells exposed to culture medium SDS

served as positive control Data represent mean ± SD, n=6 70

3.3 Uptake of fWGA by (A) Caco-2, (B) HT-29 and (C) CCD-18Co cells

as a function of incubation time (Data represent mean ± SD, n=4) 72

3.4 Uptake of fWGA by the Caco-2, HT-29 and CCD-18Co cells when

exposed to fWGA loading concentration of (A) 20 μg/ml and (B) 50 μg/ml

(Data represent mean ± SD, n=4) 74

trypan blue post-uptake, which quenched extracellular fluorescence 76

4.1 Activation of PLGA surface carboxyl groups by

1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride(EDAC)

for subsequent surface conjugation with WGA 92

4.2 (A) TEM (magnification 100,000x) and (B) SEM

(magnification of 35,000x) micrographs of WNP 109

4.3 In vitro release profiles of paclitaxel from WNP into PBS, pH 7.4, 37°C

(Mean ± SD, n=3) 110

4.4 In vitro cytotoxicity profile of blank WGA-conjugated PLGA

nanoparticles against colon cells (a) positive and negative control;

(b) Caco-2 cells; (c) HT-29 cells; (d) CCD-18Co cells

(Mean ± SC, n = 6) 111

4.5 Uptake of fWN by Caco-2, HT-29 and CCD-18Co cells as a function of

incubation time at loading concentration of 1.25 mg/ml (Mean ± SD, n = 3) 114 4.6 Uptake of fWN as a function of loading concentration by Caco-2,

HT-29 and CCD-18Co cells over an incubation period of 2h

(Mean ± SD, n = 3) 115

4.7 Uptake of fBSA-conjugated PLGA nanoparticles as a function of incubation

time at loading concentration of 1.25 mg/ml (Mean ± SD, n = 3) 115

4.8 In vitro cytotoxicity profile of paclitaxel against (a) Caco-2 cells;

(b) HT-29 cells; (c) CCD-18Co cells (Mean ± SD, n = 6) 116

4.9 In vitro cytotoxicity profiles of P/CreEL, , PNP and WNP against (a)

Caco-2 cells; (b) HT-29 cells; (c) CCD-18Co cells as a function of

incubation time and formulation concentration (Mean ± SD, n = 6) 118

4.10 Cellular uptake of paclitaxel after 2h exposure to the WNP, PNP and

P/CreEL formulations and intracellular retention of paclitaxel following

post-uptake incubation of the cells with fresh medium (a) Caco-2; (b) HT-29;

(c) CCD-18Co cells Data represent mean ± SD, n = 3 122

4.11 Confocal images of (a) Caco-2; (b) HT-29 cells incubated with

1.0 mg/ml of fluorescent WNP for 1hbefore and after TB treatment 124

4.13 Typical microscopic images of Caco-2 cells following 4 and 24h incubation

with WNP, PNP and P/CreEL formulations Cell nuclei were stained with

Hoechst 33342 127

4.14 Histograms showing the cell cycle distribution of Caco-2 cells after 4h

exposure to the paclitaxel formulations (a) Control; (b) WNP; (c) PNP;

(d) P/CreEL 128

4.15 Histograms showing the cell cycle distribution of Caco-2 cells after 24h

exposure to the paclitaxel formulations (a) Control; (b) WNP; (c) PNP;

(d) P/CreEL 129

4.16 Quantitative analysis of the cell cycle distribution of Caco-2 cells

co-cultured with paclitaxel formulations for (a) 4h and (b) 24h CON

(Control cells) 130

4.17 Typical images of Caco-2 cells showing the intracellular trafficking of

WNP following incubation of the cells with the formulation for various

time periods WNP nanoparticle has green fluorescence, cell nuclear is blue,

and the overlap of nanoparticle and lysotracker® fluorescence (red) is shown

as yellow 132

4.18 Typical confocal image of Caco-2 cells pre-treated with unlabelled

WGA and then incubated with fWNP for 3h 135

5.1 Alcian blue and PAS staining of mucin in LS174T cells following

(a) 6 days culture and (b) 3 days culture Cells were observed at

magnification of 10× 157

5.2 Lectin blot analysis for WGA-recognizable proteins among the (a) cell

membrane proteins, and (b) intracellular proteins in the LS174T cells

Lane 1 - 3 days of culture; lane 2 - 6 days of culture 158

5.3 In vitro cytotoxicity profiles of WGA against the LS174T cells as a function

of exposure time WGA was applied at loading concentrations of 0.1, 1, 5, 10,

50, 100 and 200 μg/ml Cell viability determined by the MTT assay was

expressed as a percent of that obtained for cells exposed to culture medium

Data represent mean ± SD, n=6 159

5.4 Uptake of fWGA by LS174T cells as a function of incubation time (Data

represent mean ± SD, n=4) 160

5.5 In vitro cytotoxicity of P/CreEL, PNP and WNP against LS174T cells (n = 6) 161

5.7 Fluorescence recovery curves of fWNP and fWGA in PBS solution 164

5.8 Fluorescence recovery curves of fWNP and fBSA-NP in the surface

mucin layer of LS174T cells 164 5.9 Confocal images of LS174T cells incubated with 1.0 mg/ml of fluorescent

fWNP for 1h; (a) before trypan blue treatment, (b) after incubation for 3

min with 0.2 mg/ml of trypan blue, and (c) focus on mucin layer 166

ATCC American Type Culture Collection

cm centimeter

° C Celsius degree

Da dalton

DMSO dimethyl sulfoxide

EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDTA ethylenediaminetetraacetic acid

FBS fetal bovine serum

FITC fluorescein isothiocyanate

fWGA FITC-labeled wheat germ agglutinin

fWNP fWGA-conjugated paclitaxel-loaded PLGA nanoparticles FRAP Fluorescence recovery after photobleaching

h hour

s second

HBSS Hanks balanced salt solution

HEPES N-2-hydroxyethylpiperazine-N′-2-ethanosulfonic acid

HPLC high performance liquid chromatography

IC50 50% growth inhibition concentration

IPM isopropyl myristate

MES 2-(N-morpholino)ethanesulfonic acid

MEM minimal essential medium

P/CreEL conventional paclitaxel formulation

PLGA poly(D,L-lactic-co-glycolic acid)

pH the negative logarithm of hydrogen-ion concentration

PI propidium iodide

PNP paclitaxel-loaded PLGA nanoparticles

PTA phosphotungstic acid

PVA polyvinyl alcohol

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM scanning electron microscopy

TEM transmission electron microscopy

WGA wheat germ agglutinin

WNP WGA-conjugated paclitaxel-loaded PLGA nanoparticles

UV ultraviolet

V volt

intracellular delivery of paclitaxel to Colon cancer cells Wang CX, Ho PC, LY Lim (submitted)

2 The effect of mucin of LS174T cells on the uptake of WGA-conjugated PLGA

nanoparticles Wang CX, Ho PC, LY Lim (in preparation)

3 The cytotoxicity and uptake of wheat germ agglutinin on colon cell lines, Wang

CX, Ho PC, LY Lim, Abstract for American Association of Pharmaceutical Scientists Annual Meeting, 11 – 15 November 2007, San Diego, USA

4 Loratadine transdermal patches Liu Yuling, Wang Chunxia Patent CN03134651.0 (China)

5 Nonlogarithmic titration method for the determination of dissociation constant of

loratadine, Chinese Pharmaceutical jounal, Wang Chunxia, Liu Yuling 2003,

38(11): 860-861

6 The New Development of Transdermal Drug Delivery System, Acta Pharmaceutica Sinica, Wang Chunxia, Liu Yuling, 2002,37(12): 999-1002

Chapter 1 Introduction

1.1 Chemotherapy for colon cancer

Successful systemic cancer chemotherapy, first developed in 1946, was highlighted in

a review by Gilman and Philips (Gilman & Philips, 1946) which also marked the beginning of modern cancer chemotherapy (Pratt et al., 1994) Chemotherapy is often needed for colon cancer patients after surgical removal of the tumor in order to reduce the risk of recurrence Various anticancer agents are used, depending on the stages of colon cancer and patient condition While most are highly effective, anticancer agents are also potent agents which can cause serious side effects Unfortunately, the systemic administration of most chemotherapeutic drugs is non-discriminative, causing both cancerous and healthy tissues to be concomitantly exposed to the drugs, resulting in high mortality and morbidity This provides the impetus to develop drug delivery platforms capable of targeting the delivery of anticancer agents to cancerous colonic cells

1.1.1 Colon cancer

Anatomically, the colon, also known as the large intestine or large bowel, is part of the intestine that extends from the cecum to the rectum Colon cancer refers to cancer that is located in the colon or rectum, and it is sometimes called “colorectal cancer”

In the United States, colon cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related deaths (American Cancer Society Cancer

Facts & Figures, Atlanta, 2008) In Asia, colon cancer is also the third commonest cancer disease, but its incidences in many Asian countries are rapidly increasing (Sung, 2007) In Singapore, colon cancer has overtaken lung cancer as the commonest cancer and is the second commonest cause of cancer-related deaths

of patients (Abraham et al., 2005) However, relapse following surgery is a major problem, and is often the cause of death If surgery reveals that the cancer has spread

to other organs or tissues, chemotherapy alone or combination with other modalities is usually prescribed (Oehler & Ciernik, 2006)

Cancer chemotherapy aims to shrink the tumor size, slow tumor growth and/or reduce the likelihood of metastasis developing In most instances, the primary focus is on

killing rapidly dividing cells Unfortunately, some normal cells also have a high rate

of division e.g cells in the bone marrow and hair follicles, and most chemotherapeutic delivery systems are unable to differentiate between rapidly dividing cancerous cells from other fast proliferating normal cells Consequently, cancer chemotherapy is often associated with serious side effects To circumvent this drawback, drug delivery targeted specifically to colon cancer cells is desired

1.1.2 Anticancer agents

The first clinically effective anticancer drug developed was nitrogen mustard, an agent initially developed as a war gas Alkylating drugs based on nitrogen mustard were subsequently synthesized and developed for cancer chemotherapy These drugs, like nitrogen mustard, are DNA cross-linkers and they prevent DNA synthesis in cells Many anticancer drugs with different mechanisms of action have since been developed (Cavalli et al., 2000), and they include alkylating and intercalating agents,

as well as topoisomerase inhibitors, which cause direct DNA damage; antimetabolites that interfere with DNA synthesis by inhibiting key enzymes in purine or pyrimidine synthesis, or by misincorporation into the DNA molecule to cause strand breaks or premature chain termination; antibiotic cytotoxic agents that alkylate DNA or inhibit topoisomerase II or both; spindle poisons that bind to tubulin to inhibit tubulin polymerization or microtubule depolymerization

At the mechanistic level, cellular microtubules-interacting agents are one of the most attractive and promising approaches for cancer chemotherapy Microtubules are filamentous polymers that constitute one of the major components of the cytoskeleton They are a superb target because of their important function in mitosis and cell division Table 1.1 shows a list of microtubule-targeted drugs currently in clinical use

or under clinical development

Table 1.1 Microtubule-targeted drugs, their binding sites on tubulin, and their stages

of clinical development (Jordan & Kamath, 2007)

Binding

Domain Drugs Therapeutic uses Stage of clinical development Vinca

Domain Vinblastine (Velban) testicular germ cell cancer Hodgkin’s disease,

In clinical use, many combination trials in progress Vincristine (Oncovin) Leukemia, lymphomas In clinical use, many combination trials Vinorelbine (Navelbine) Solid tumors, lymphomas, lung caner clinical trials, Phases I-III, In clinical use, many

single and combination Vinflunine lung cancer, breast cancer Bladder, non-small cell Phase III

Cryptophycin 52 advanced ovarian caner Platinum-resistant Phase II

Dolastatins (TZT-1027, Tasidotin) Vascular targeting agents Phase I, II

Maytansinoids (conjugated to humanized monoclonal antibody)

Colon, lung, pancreas Phase II

Colchicine

Non-neoplastic diseases (gout, familial Mediterranean fever), also actinic keratoses

Appears to have failed most cancer trials, presumably due to toxicity

Combretastatins (AVE8062 CA-1-P, CA-4-P (combretastatin A4 phosphate), N-acetylcolchicinol-O-pho sphate, NPI-2358, ZD6126)

Vascular –disrupting

2-Methoxyestradiol glioblastoma multiforme Multiple myeloma,

carcinoid, prostate, breast Phase I, II Methoxybenzene-sulfonam

ide (ABT-751, E7010) vascular-disrupting agent Solid tumors, Phase I, II Diketopiperazine

(NPI-2358) Vascular-disrupting agent Phase I

Taxane Site

Paclitaxel and analogs (Taxol, TL00139, XRP9881, XRP6258, BMS844476, BMS188797,

TPI287)

Ovarian, breast, lung tumors, Kaposi’s sarcoma, trials with numerous other

tumors

In clinical use and in combination and single drug trials, Phase I-III

Docetaxel (Taxotere) Prostate, brain, breast, and lung tumors Phase I-III

Epothilone (Ixabepilone, epothilones B (patupilone),

D, ZK-EPO, KOS-862, KOS-1584)

Taxol-resistant tumors Phases I, II, III

Discodermolide Synergistic with taxanes Phase I Other

Microtubule

Binding

Sites

Estramustine Prostate, often in combination

Phases I, II, III, in combinations with taxanes, epothilones, and Vinca alkaloids Arsenic trioxide (trisenox) Vascular-disruptive agent

There are two distinct groups of microtubule-interacting agents; the first group inhibits the assembly of tubulin heterodimers into microtubule polymers while the other stabilizes microtubules under normally destabilizing conditions to prevent their disassembly The latter, of which paclitaxel is a representative compound, will be the focus of this project A variety of natural products have been discovered over the last decade to inhibit the proliferation of human cancer cells through a paclitaxel-like

mechanism These compounds represent a whole new set of structurally varied lead compounds for anticancer chemotherapy

1.1.3 Paclitaxel

Paclitaxel, a diterpenoid derivative, was first discovered at the Research Triangle Institute (RTI) in 1967 when Drs Monroe E Wall and Mansukh C Wani isolated the

compound from the bark of the Pacific yew tree, Taxus brevifolia, and noted its

antitumor activity in a broad range of rodent tumors By 1970, the two scientists had determined the extremely complex structure of paclitaxel (Hennenfent & Govindan, 2006) (Figure 1.1)

Figure 1.1 Molecular structure of paclitaxel (Singla et al., 2002)

Since then, paclitaxel has been found to be effective against a wide variety of tumors, including refractory ovarian cancer, breast cancer, lung cancer, colon cancer, head and neck carcinomas, and acute leukemia (McGuire & Rowinsky, 1995) It works by interfering with the normal function of microtubules in cells Paclitaxel binds specifically to the β subunit of tubulin, the "building block" of microtubules (Horwitz, 1994), and the resultant complex does not have the ability to disassemble This adversely affects cell function because the shortening and lengthening of microtubules (termed dynamic instability) is necessary for the transport of other cellular components For example, during mitosis, microtubules position the chromosomes during their replication and subsequent separation into the two daughter-cell nuclei (Horwitz, 1994; Wang et al., 2005) Consequently, the stabilization of microtubules prevents cancer cells from restructuring their cytoskeleton in a flexible manner and hinders their aggressive division (Wang et al., 2005) Although normal cells can also

be adversely affected, cancer cells are far more susceptible to paclitaxel treatment because of their much faster rate of cell division Further research has indicated that paclitaxel also induces programmed cell death (apoptosis) in cancer cells by binding

to a protein called Bcl-2 (B-cell leukemia 2), which is responsible for arresting apoptosis (Akay et al., 2004)

Despite its effectiveness, paclitaxel is not without disadvantages One of the major clinical problems associated with paclitaxel is its extreme hydrophobicity, and thus very low solubility in water (~6-30 mg/ml) The clinical formulation, Taxol®, contains paclitaxel (6 mg/ml) dissolved in a 50:50 v/v mixture of the surfactant, Cremophor

EL® (polyoxyethylated castor oil), and dehydrated ethanol (Straubinger, 1995; Hennenfent & Govindan, 2006) Cremophor EL® has been found to contribute to serious side-effects, e.g severe hypersensitivity reactions, and is incompatible with common PVC intravenous administration sets To avoid the hypersensitivity reactions, patients have to be pretreated with corticosteroids (e.g dexamethasone), diphenhydramine and H2-receptor antagonist (e.g cimetidine, ranitidine) before Taxol® administration (Singla et al., 2002) Precipitation of paclitaxel is also an issue when the formulation has to be diluted for clinical use (Pfeifer et al., 1993)

Though paclitaxel is a very potent anticancer agent against colon cancer cells, its efficacy is limited because of low solubility and poor specificity against cancer cells

In order to improve the bioavailability, resolve the intractable solubility and minimize the cytotoxicity and adverse side effects associated with paclitaxel therapy, a targeted drug delivery system needs to be developed Many modified formulations and more water-soluble analogues of paclitaxel have been investigated (Ibrahim et al., 2002; Kim et al., 2004; Soepenberg et al., 2004) Among the methods proposed,

nanoparticles of biodegradable polymers are promising in resolving the twin issues of the adjuvant problem and the controlled and targeted delivery of the drug (Serpe et al., 2004; Weissenbock et al., 2004; Musumeci et al., 2006) A nanoparticle formulation

of paclitaxel bound to albumin (ABI-007) has been approved for the treatment of metastatic breast cancer (Gradishar et al., 2005) Paclitaxel incorporated into polymer nanoparticles has demonstrated a significant specificity of action mainly because of changes in its tissue distribution and pharmacokinetics (Couvreur et al., 1980) These changes, directed by the nanoparticle pharmacokinetics, have the potential to improve the therapeutic efficacy of paclitaxel with concomitant reduction of its side effects and toxicity In this project, paclitaxel was used as a model drug to develop a nanoparticle delivery system with WGA as a targeting moiety with the view to resolve current clinical issues associated with paclitaxel therapy

1.1.4 Targeted delivery of anticancer agents

Much effort has been expended to improve the selectivity of cancer chemotherapeutic agents, and significant improvement in patient survival has been achieved in recent years Nevertheless, the developments of novel selective anticancer agents and new ways of delivering both old and new agents are possibly the most important goals of modern anticancer research

Targeted cancer chemotherapy aims to direct adequate concentration of the chosen agent to tumor cells while affecting as few healthy cells as possible In principle, this can be achieved by passive or active targeting Passive targeting exploits the enhanced permeability and retention (EPR) characteristics of tumor vessels Rapidly growing tumors develop extensive vasculatures to meet their requirement for nutrient supply and waste disposal, but the blood vessels are abnormally hyper-permeable, with defective architecture and impaired lymphatic drainage (Nie et al., 2007) Circulating macromolecular drugs or particulate delivery systems that have difficulty permeating normal blood vessels can extravasate through such tumor blood vessels, and they become entrapped due to the impaired lymphatic drainage in tumor tissues Consequently, the EPR effect can be applied to facilitate the selective accumulation of

an appropriately designed drug delivery system at a tumor site To achieve efficient accumulation, the delivery system must also avoid systemic clearance by the reticuloendothelial system (RES), usually achieved by controlling the size and surface properties of the delivery systems (Gref et al., 1994) To avoid RES uptake, a hydrophilic surface and small particle size under 100 nm are the most often mentioned requirements (Ameller et al., 2003; Owens & Peppas, 2006) Active targeting, on the other hand, is often achieved by exploiting the differences in membrane biochemistry between cancer and normal cells Active targeting of a drug

to cancer cells may involve the conjugation of tissue- or cell-selective ligands

(Brannon-Peppas & Blanchette, 2004) that bind specifically with receptors on the surface of tumor cells, examples of which include lectin-carbohydrate and antibody-antigen interactions (Allen, 2002) Selectivity is ensured by choosing ligands that bind to antigens or receptors that are either uniquely expressed or over-expressed on the target cells compared to normal tissues To increase the payload to be delivered to the tumor site, the drug may be concentrated in a carrier, e.g nanoparticles, which is then conjugated with the targeting ligand (Fonseca et al., 2002; Garber, 2004)

Since drug administration routes and the mechanisms of drug delivery are different, specific bioconjugates have to be designed according to the delivery pathways and the characteristics of the tissues involved The possibility of targeting drugs specifically

to the colon has been shown to be feasible (Chourasia & Jain, 2003), and ligands such

as lectins may have the potential to achieve this goal (Dalla Pellegrina et al., 2005) Wheat germ agglutinin (WGA) is an example of plant lectins that binds specifically to the N-acetyl-D-glucosamine sugar residues expressed by cancer cells and normal gastrointestinal epithelial cells This specific characteristic may allow WGA to be applied as a ligand for targeting drug delivery to colon cells Moreover, the binding of WGA to the gastrointestinal epithelial cells could prolong the retention time, result in high local concentration and improve the absorption profile of the drug Selective

binding to cancer cells that expressed the WGA-binding glycoprotein would also facilitate the uptake of the WGA-conjugated delivery system into the cells

The colonic mucosa, like other exposed epithelial surfaces, is covered by a mucus gel layer which protects the underlying epithelium against mechanical damage, biological agents and chemical irritants Mucus is a complex mixture of large glycoproteins (mucins), water, electrolytes, sloughed epithelial cells, secretory immunoglobulin A (Ig A), lysozyme, lactoferrin and α1-antitrypsin (Macfarlane et al., 2005) It forms a dynamic viscoelastic gel with high water content (95%) and specific physical properties, which allows the gel to flow An important function of the mucus gel is to provide a medium for interaction between the lumen and the mucosal cells, and it has been referred to as an “unstirred aqueous layer” (Aksoy & Akinci, 2004) The macromolecular glycoprotein component (mucin) is responsible for the viscoelastic nature of mucus In the intestinal mucus, the glycoproteins contain 77.5% of carbohydrates which comprise N-acetyl-galactosamine, N-acetyl-glucosamine, galactose, fucose and sialic acid at a dry weight molar ratio of 1.0:0.6:0.7:0.3:0.5 (Moghimi et al., 2001) The high viscosity of mucus can hinder drug diffusion and, consequently, drug absorption by underlying cells

Most common malignant tumors of the large intestine are characterized by mucin production The adenocarcinoma, a major colon tumor, forms moderate to well-differentiated glands that secrete variable amounts of mucin The most aggressive tumor of the colon, the mucinous carcinoma, is associated with abundant mucin secretion (Aksoy & Akinci, 2004), which may impede normal drug delivery to the cancer cells In this respect, mucoadhesive drug delivery systems that bind with mucin may prove advantageous, as they could improve contact between drug and intestinal cells, as well as prolong the residence time

1.2 Polymeric nanoparticles

1.2.1 Nanoparticulate systems for drug delivery

Nanoparticles are engineered submicron-sized systems that range in size from a few nanometers to several hundred nanometers depending on their intended use (Haley & Frenkel, 2008) A variety of organic and inorganic materials, including polymers, lipids, ceramic and metals, have been used to construct nanoparticles (Yezhelyev et al., 2006) Most inorganic nanoparticles have a central core (usually metallic) and a protective organic surface coating Organic nanoparticles include liposomes and other lipid-based carriers, polymeric nanoparticles, micelles and various ligand-targeted products Structurally, nanoparticles have also been classified as dendrimers, micelles,

nanospheres, nanocapsules, liposomes, fullerenes and nanotubes Based on their manufacturing methods and materials used, the size and shape of nanoparticles vary

Therapeutic drugs may be incorporated into nanoparticles by surface attachment or encapsulation (Haley & Frenkel, 2008) Nanoparticulate drug delivery systems are highly versatile Drug payloads range from small molecular weight drugs to macromolecules, from highly water-soluble agents to strongly hydrophobic drugs The method of delivery may vary from the simple, localized delivery using a catheter-based approach (Song et al., 1998) to sophisticated targeted delivery whereby the conjugation of biospecific ligand onto the nanoparticle surface could direct drug delivery to the tissue of interest (Moghimi et al., 2001) In addition, the small particle size of nanoparticles yields a high surface area per unit weight ratio that can greatly facilitate drug dissolution and absorption in the gastrointestinal fluids Nanoparticulate systems have been demonstrated to improve drug bioavailability (de Salamanca et al., 2006), facilitate drug solubilization (Merisko-Liversidge et al., 2003), sustain drug effect in target tissues (Moghimi et al., 2001) and improve the stability of therapeutic agents (Chavanpatil et al., 2006) Since the latter half of the 1980s, nanoparticles have been studied as carriers for drug delivery to challenge many diseases, including cancer, HIV, and diabetes (Yih & Al-Fandi, 2006) Much of the research has concentrated on improving the bioavailability of drugs with poor

absorption characteristics and providing controlled release of drugs Table 1.2 gives

examples of the many different types of nanoparticulate systems currently under

various stages of clinical investigation (Wang et al., 2008)

Tabel 1.2 Examples of polymeric nanoparticles in clinical development

Albumin-paclitaxel Abraxane or ABI-007 Market Metastatic breast cancer

Various cancers, particularly non-small-cell lung cancer;

ovarian cancer PEG-aspartic acid-doxorubicin micelle NK911 Phase I Pancreatic cancer

HPMA copolymer-doxorubicin PK1; FCE28068 Phase II Various cancers, particularly lung and breast cancer

HPMA

copolymer-doxorubicin-galactosamine PK2; FCE28069 Phase I/II Particularly hepatocellular carcinoma

HPMA copolymer-paclitaxel PNU166945 Phase I Various cancers

HPMA copolymer-camptothecin MAG-CPT Phase I Various cancers

HPMA copolymer-platinate AP5280 Phase I/II Various cancers

HPMA copolymer-DACH-Platinate AP5346 Phase I/II Various cancers

Dextran-doxorubicin AD-70, DOX-OXD Phase I Various cancers

Modified dextran-camptothecin DE-310 Phase I/II Various cancers

Abbreviations: PEG, polyethylene glycol; DACH, diaminocyclohexane; HPMA,

N-(2-hydroxypropyl)methacrylamide

Therapeutic agents of interest are incorporated into polymer nanoparticles either by physical entrapment within the polymeric matrix or by surface adsorption or conjugation The size and surface properties of the nanoparticles determine their fate

in the human body Unless there is intended drug delivery to the RES, the size and surface of the nanoparticles must be designed to avoid RES clearance Nanoparticles smaller than 100 nm in diameter have been found advantageous in this respect (Munshi et al., 1997) So is the coating of nanoparticles with the hydrophilic polymer, polyethylene glycol (PEG) (Laverman et al., 2001) Targeted drug delivery is realized

by surface conjugation with a biospecific ligand, which may also favorably modify the intracellular disposition of the nanoparticles Biocompatible, hydrophilic or hydrophobic polymer nanoparticles with surface-pendant amine, carboxyl or aldehyde groups have been fabricated for further bio-conjugation A wide variety of ligands, such as folic acid (Das et al., 2008), antibody (Natarajan et al., 2008), and aptamers (Farokhzad et al., 2004) have been used for surface modification of polymeric nanoparticles to impart cancer cell targeting capability

Polymers employed for nanoparticle fabrication have included synthetic polymers, such as Poly (D, L-lactic-co-glycolic acid) (PLGA), polyacrylates and polycaprolactones, and natural polymers (Wong et al., 2007) In particular, the application of polymer nanoparticles in oncology has grown greatly with the advent of

biodegradable polymers (Raghuvanshi et al., 2002; Kreuter et al., 2003) Biodegradable polymers are macromolecular materials capable of being degraded into simpler products through chemical or enzyme-catalyzed hydrolysis in the body Biodegradable and biocompatible polymers as drug carriers are desirable to minimize toxicity and avoid the requirement to surgically remove the spent carriers For these reasons, nanoparticles made of biodegradable materials are often fabricated to provide sustained drug release within the target site (Westedt et al., 2007; Yang et al., 2007)

A good example of a biodegradable and biocompatible polymer is PLGA, an

FDA-approved biodegradable and biocompatible polymer for biomedical applications

1.2.2 PLGA nanoparticles

PLGA is a synthetic copolymer of lactic acid and glycolic acid and is one of the most widely used FDA-approved biodegradable polymers for controlled release drug delivery systems The biodegradation of PLGA occurs through a hydrolytic chain

cleavage mechanism In vivo, PLGA undergoes chemical hydrolysis as well as

enzymatic cleavage of its backbone ester linkages (Astete & Sabliov, 2006) to form the biologically compatible moieties, lactic acid and glycolic acid The degradation products are subsequently eliminated from the body as carbon dioxide and water by the tricarboxylic acid cycle (Jalil & Nixon, 1990; Anderson & Shive, 1997) The

chemical structure of PLGA is illustrated in Figure 1.2, where “m” and “n” refer to the relative amounts of lactide and glycolide units, respectively, in a specific PLGA copolymer The composition of PLGA can be varied by modifying the chain length (molecular weight), as well as the ratio of lactic to glycolic acid monomers in the polymer chain This flexibility of composition is advantageous as it can be manipulated to yield appropriate physical properties for a particular application For

example, the in vivo degradation rate of PLGA can be tailored by controlling the ratio

of “m” and “n”, with slower degradation rates observed for polymers with higher m/n ratios (Lin et al., 2000)

Figure 1.2 Chemical structure of PLGA and its degradation products (Taluja et al.,

PLGA has a long history of safe use as surgical sutures and implants, and it is applied

in at least 12 different marketed products from 10 different companies worldwide (Avgoustakis, 2004) PLGA is used not only as a resorbable suture material and a scaffold for tissue engineering, but also in drug delivery (Ignatius & Claes, 1996; Day

et al., 2005) PLGA delivery platforms have been developed for the sustained and targeted delivery of plasmid DNA (Abbas et al., 2008); recombinant HIV envelope (env) protein (Moore et al., 1995); hormones (Sun et al., 2008) and anticancer agents (Gryparis et al., 2007; McCarron et al., 2008) PLGA nanoparticles have been formulated as colloidal carrier systems to improve drug efficacy (Brigger et al., 2002; Sahoo et al., 2004; Fukumori & Ichikawa, 2006) Drug release from PLGA nanoparticles is controllable through the rate of drug diffusion in the polymer matrix

and/or degradation of the polymer matrix (Hariharan et al., 2006)

1.2.2.1 Preparation

Preparation of nanoparticles is frequently based on the use of dispersed systems in which solid or liquid phases are dispersed in fluid media to constitute embryos of the final particles Single and multiple emulsion systems have been used to encapsulate drugs into polymeric particles Normally, an organic solvent is required to dissolve